CHIT3 | Chitinase 3
AS15 2888 | clonality: polyclonal | host: rabbit | reactivity:
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|Expected | apparent MW||
|Predicted reactivity||Musa paradisiaca, Zea mays|
|Not reactive in||
no confirmed exceptions from predicted reactivity known in the moment
to be added when available
|Selected references||to be added when available, antibody released in June 2015|
Total protein from Oryza sativa rice (CV. 9311) flag leaf at the flowering stage was ground into a fine powder in liquid nitrogen. An 800 ul aliquot of extraction buffer: 62.5 mM TRIS-HCl (pH 7.4), 10% glycerol, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5% (v/v) b-mercaptoethanol] was added to each 300 mg powder sample. The mixture was vortexed and then chilled on ice for 10 min. Samples were centrifuged at 12,000 rpm for 10 min. at 4℃, and the supernatant was collected and stored at –70℃. The protein concentrations of the rice samples were determined using the Bradford method (Bradford, 1976). 20 µg of protein was separated on 12 % SDS-PAGE and blotted 1h to PVDF.
Blots were blocked with for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:10 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturer’s instructions. Exposure time was 30 sec.
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